Method for constructing transgenic economic seaweeds

ABSTRACT

This invention relates to marine algae, and more particularly, to a method for producing improved seaweed strains by genetic engineering. The vector for transformation were constructed by inserting the high-plant or algae-derived promoters upstream of foreign reporter genes or such cassettes that functional genes are fused with antibiotics or herbicide-resistant genes. The genetic seaweed was generated by natural development process by recombinated plasmid DNA introduction to seaweed spore with Biolostics as transformation methods. Introduced traits of antibiotics or herbicide-resistance were used to select the transgenic seaweed individuals when foreign functional genes are transformed. Stable transformation could be obtained following this invention.

FIELD OF THE INVENTION

This invention relates to marine algae, and more particularly, to themethod for producing improved seaweed strains by genetic engineering.

BACKGROUND OF THE INVENTION

Phycocolloids are natural gums produced by and extracted from marinealgae. The three principal types of commercially valuable phycocolloidsare agar, carrageenan and alginate. By far the seaweeds which have beensuccessfully cultivated include red algae (Porphyra, Gracilaria,Gelidium, Euchema), green algae (Ulva) and brown algae (Laminaria,Undaria). The global cultivation areas for seaweeds are 3 million mu,with annual production more than 6.35 million tons (wet weight), most ofwhich were utilized to extract the phycocolloids, except a small amountas nutrition food. In recent years, due to quickly-increased demands tothe phycocolloids production and quality improvement, the genetictransformation techniques were introduced to the seaweed researchfields, with purpose to directionally improve the seaweeds traits, alsothe great potential for expression of high-value products has beensuggested.

To date, most of the well-developed techniques to generate transgenicplant are employed for high plants, which include:

1. Transformation methods: Agrobactarium Ti plasmid-mediatedtransformation, electroporation to protoplast, and Biolistics toexplant; 2. Vector element: high plant-derived promoters (i.e. CaMV35S)have been used widely to drive the expression of foreign genes; 3.Transformation recipient and plant regeneration: to take advantage ofthe totipotency, that is to regenerate protoplast, or induce the callusformation and further differentiation to regenerate new plant; 4.Selectable marker: Npt II gene was used most widely as selectable markerthat, transgenic plant could be obtained by selection with kanamycin orneomycin.

Seaweeds are low plants that live in marine environment, they are ofsignificant difference which including: 1. Transformation methods: nowell-developed methods like Agrobactarium Ti plasmid-mediatedtransformation to high plant; 2. Vector element: no seaweed-derivedpromoter has been obtained; 3. Transformation recipient and plantregeneration: there's significant difference on research extent betweenspecies, protoplast regeneration was easy in some genus like Porphyra,but very hard in others like Laminaria and Undaria, as well as lowefficiency for callus induction; 4. Selectable marker: sensitiveantibiotics types and sensitivity extent maybe different between seaweedand high plant.

By far most researches on seaweed transformation have referred thetechniques applied in high plant, those are to transfer the seaweedexplants or protoplast as transformation recipient. Only transientexpression has been obtained, and no expression was detected inregenerated plant, even the transient transformation efficiency is lowsince only high plant-derived promoters (e.g. CaMV35S) are available; nosensitivity test on seaweed to antibiotics have been done yet.

SUMMARY OF THE INVENTION

Aiming to solve such difficulties referenced above, this inventionprovides the method for producing improved seaweed strains by geneticengineering with high efficiency and stable expression, taking advantageof seaweed life cycle characters.

In order to realize the purposes above-mentioned, the technical programof this invention is as follows. The vector for transformation wereconstructed by inserting the high-plant or algae-derived promotersupstream of foreign reporter genes or such cassettes that functionalgenes fused with antibiotics or herbicide-resistant genes. The seaweedspore was employed as transformation recipient and bombed withrecombinated plasmid DNA by Biolostics, thus the genetic seaweed couldbe generated by natural development process. Introduced traits ofantibiotics or herbicide-resistance were adopted to select thetransgenic seaweed individuals when foreign functional genes aretransformed, therefore, to obtain further the foreign gene products(e.g. expressed vaccine, defensin, antibody, medical protein).

Wherein the said foreign functional genes are those encodingphycobillisome, growth factor, antibody, vaccine, defensin orantibacterial peptide and so on; said antibiotics or herbicide-resistantgenes are cat, hpt or bar; said antibiotics are chloramphenicol orhygromycin; said herbicide is Basta; said promoters are SV40 promoter,CaMV35S promoter, ubiquitin promoter or algae-derived fcp promoter andso on; said selectable marker-selectable reagent cassettes arecat-chloramphenicol, hpt-hygromycin or bar-Basta.

Compared with present techniques employed in seaweeds, this invention isan entire innovation which addressed significant improvement as bellows:

1. In terms of transformation methods, although Biolistics is auniversal penetrating-cell wall method with a wide range of availabletarget cell, this invention is the first one to offer the seaweed sporesas transformation recipient in the field.

2. In terms of vector element, the experiments of this invention shownthat available promoters for seaweed has been explored a lot among thosederived from both high plant and algae.

3. In terms of transformation recipient, the second original creation ofthis invention is to obtain non-chimeric expression of transgenic plantby using seaweed spore as transformation recipient. For most cultivatedseaweed, pure germ plasm bank have been set up by using theirmicroscopic life stage, such as male and female gametophytes inLaminaria and Undaria, filamentous sporophytes in Prophyra, andtechniques to generate new plant from seaweed spore have been welldeveloped, which contribute reliable materials for spore-mediatedtransformation in seaweed.

4. In terms of selectable marker, research results of this inventionshown that Laminaria and Undaria are sensitive to chloramphenicol, muchmore to hygromycin, and most sensitive to Basta, thus three selectablemarker-selectable reagent cassettes are applicable includingcat-chloramphenicol, hpt-hygromycin and bar-Basta.

5. Stable expression of foreign gene could be obtained by thisinvention, in the case of lacZ reporter gene transformation to Laminariamale gametophytes, the transformation efficiency in zygote sporophytesis up to 50%, with expression efficiency up to 30%.

DESCRIPTION OF THE INVETION IN DETAIL

The invention will be further described in detail with reference todrawings attached and examples below.

EXAMPLE 1 Invention Effective to Laminaria Female Gametophytes withReporter Gene Transformation

A vector contains GUS reporter gene driven by fcp promoter (from marinediotam-derived fcp gene encoding fucoxanthin-chlorophyll a/c bindingprotein) was introduced to Laminaria female gametophytes by Biolistics,resulting in stable expression of GUS gene in parthenogeneticsporophytes, detailed description is as below:

1. Preparation of Micro-Particle for Biolistics:

To weight 60 mg golden powder (accessory to Biolistics, Bio-Rad, USA,1.0 μm in diameter) and add 1 ml ethanol, vortex for 1 min followed bybrief centrifuge at 10000 rpm for 10 sec. To remove the supernatant,wash the golden particle by adding 1 ml autoclaved water to resuspendand centrifuging to remove the supernatant, repeat the wash process for3 times, resuspend the golden particle in final 1 ml autoclaved water,aliquot into 50 μl each and store at 4° C.

Before transformation, each aliquot (50 μl) was transferred to a 1.5 mlcentrifuge tube, and added into 5 μl DNA (1 μg/μl), 50 μl CaCl₂ (2.5mol/L), 20 μl spermidine (0.1 mol/L) in order during continuousvortexing, last to vortex for 3 min, centrifuge at 10000 rpm for 10 secand remove the supernatant, wash the pellet by using 250 μl ethanol for2 times, resuspend the golden particle in 60 μl ethanol.

2. Transformation to Laminaria Female Gametophytes:

In 1-2 days before transformation, to gently grind the Laminaria femalegametophytes by using 2 pieces of autoclaved glass slides, to separatethe filamentous to a extent that no fragments more than 5 cells could bedetected under microscope. To count the cells by a hematimeter andculture the suspension in dark overnight. Round autoclaved silk sievecloth (400 mesh) was used as carrier for target cells, which are tiledas a round and thin layer with diameter in 2 cm in the middle of thecloth. Each sample containing 1.0×10⁶ female gametophyte cells wastransformed once with about 12 μl resuspension of golden particle.

Biolistics (Type: PDS1000/He) used for transformation is product ofBio-Rad Ltd. Company, USA. Both table-board and outside and insidesurface of Biolistics was sterilized with 70% ethanol, involvedaccessories including Rupture Disk, DNA Macrocarrier and Stopping Screenwhich are all rinsed in 70% ethanol for 20 min, and dried under UVlight. Biolistics parameters used for transformation are: the distancebetween target cells and Stopping Screen is 6.0 cm, vacuum is 28 (inchmercury column).

Control groups which were transformed with empty golden particle, i.e.no DNA coated, were also set up, sharing the same procedure.

3. Culture After Transformation:

After transformation, Laminaria female gametophytes from both treatmentand control group were transferred to Ferric-containing sea water media(prepared by using autoclaved sea water, containing NaNO₃ 0.71 mmol/L,KH₂PO₄O 0.032 mmol/L, Ferric citric 0.0019 mmol/L, V_(B12) 0.5 μg/L), attemperature 10.0±0.5° C., under light cycle of L:D=10 h/14 h, and theillumination degree of 100 μmol·m⁻²·s⁻¹. The same media was employed forparthenogenetic juveniles under the same condition, with media renewedevery week.

4. Detection for Integration and Expression of GUS Gene:

The in situ histo-chemical staining method was adopted to detect the GUSexpression in parthenogenetic sporophytes of Laminaria. Positive resultwas obtained by PCR, suggesting the occurrence of integration of GUSgene. Whereas no expression was detected in control group transformedwith empty golden particle without DNA coated.

In this description, foreign genes refer to those exogenous to thetarget seaweed, including both reporter gene helpful to set up thetransformation model, and functional gene encoding certain protein orpeptides with applicable utilization, such as vaccine, antibody and soon. In this example, the foreign gene is a kind of reporter gene. Thepromoter refers to a special nuclear acids sequence, which can drive theexpression of foreign gene.

EXAMPLE 2 Invention Effective to Both Female and Male LaminariaGametophytes with Reporter Gene Transformation. (the Example 1 and 2 areMethodology Proof for Spore-Mediated Transformation)

A vector contains lacZ reporter gene driven by SV40 promoter wasintroduced to both female and male Laminaria gametophytes by Biolistics,resulting in stable expression of lacZ gene in zygotic sporophytes. Allprocedures about golden particles preparation and Biolisticstransformation are as same as in Example 1. Procedures for target cellspreparation are same between female and male gametophytes. Subsequentsteps are:

3. Culture After Transformation:

After transformation, Laminaria gametophytes from both treatment andcontrol group were transferred to Ferric-containing sea water media asin example 1 under the same culture condition. To add the same amount ofnon-transformed male gametophytes to female transformed gametophytes andnon-transformed female gametophytes to male transformed gametophytesrespectively. Same media was employed for zygotic sporophytes juvenilesunder the same condition, with media renewed every week.

4. Detection for Integration and Expression of lacZ Gene:

in situ histo-chemical staining method was employed to detect the lacZexpression in zygotic sporophytes of Laminaria, positive result wasobtained in both male gametophytes-mediated and femalegametophytes-mediated treatment groups. Transformation efficiency in theformer is 50%, expression efficiency (calculated as percentage ofstained area in whole area of each positive individual) is about 30%,showing a significant enhancement compared with the latter (asTransformation efficiency 20% and expression efficiency 4-5%). There is1.9% of individuals in former group showed uniform expression, combinedwith the positive PCR results, it has suggested that the integration oflacZ gene occurred at one-cell stage.

EXAMPLE 3 Extension to Utilization, Both Functional Foreign Gene andSelectable Marker were Used

A vector contains a cassette (selectable marker cat-vaccine gene HBsAgencoding Hepatitis B surface antigen) driven by SV40 promoter wasintroduced to Laminaria male gametophytes by Biolistics, resulting instable expression of HBsAg gene in zygotic sporophytes afterchloramphenicol selection.

All the procedures about golden particles preparation, male gametophytescells preparation and Biolistics transformation are as the same as inthe Example 1. The procedures for culture after transformation are asthe same as in the Example 2. Subsequent steps are:

3. Selection by Chloramphenicol

Chloramphenicol selection was performed in autoclaved beakers by addingN-P-containing sea water media, chloramphenicol and zygotic sporophytesjuveniles reaching 2 cm in length. Chloramphenicol concentration was 20μg/ml during the first 2 days, and increased to 50 μg/ml during the3th-7th day, with media renewed everyday.

4. Detection to Integration and Exression of HBsAg Gene

The HBsAg expression in 38 samples was determined by quantified ELISAmethod, and 13 samples (34.21%) were positive, determined expressionlevel is up to 1.2 μg/(mg soluble protein), averaging 0.465 μg/(mgsoluble protein). The positive PCR result suggests the successfulintegration of HBsAg gene.

5. Application of Transgenic Kelp Expressing HBsAg

Some research has showed that there's no significant difference onvaccine dosage between vessel injection and oral administration.Injection dosage for an adult is about 15 μg, corresponding to about thesame amount of vaccine expressed in 2.5 g (fresh weight) of transgenickelp, suggesting a great application potential for transgenic seaweed byusing this invention.

Selectable marker refers to a antibiotics or herbicide resistance gene,whose expression will benefit the host cell to survive under thecorresponding antibiotics or herbicide pressure, whereas others withoutthis selectable marker will die. In this example, the functional foreigngene is vaccine HBsAg gene, and the selectable marker is cat gene.

EXAMPLE 4 Extension to Utilization, Both Functional Foreign Gene andSelectable Marker were Used

A vector contains a cassette (selectable marker hpt-defensin gene)driven by SV40 promoter was introduced to Laminaria female gametophytesby Biolistics. The positive PCR result was obtained in survivals ofparthenogenetic sporophytes after hygromycin selection.

All procedures about golden particles preparation, female gametophytescells preparation and Biolistics transformation are as the same as inthe Example 1. Hygromycin selection was performed in autoclaved beakersby adding N-P-containing sea water media, hygromycin and zygoticsporophytes juveniles reaching 1-2 cm in length. Hygromycinconcentration was 50 μg/ml, with media renewed every week.

EXAMPLE 5 Invention Effective to Undaria with Reporter GeneTransformation

A vector contains lacZ reporter gene driven by SV40 promoter wasintroduced to Undaria male gametophytes by Biolistics, resulting instable expression of lacZ gene in both male gametophytes and zygoticsporophytes.

All procedures about golden particles preparation, male gametophytescell preparation and Biolistics transformation are as the same as in theExample 1. The pocedures for culture after transformation are as thesame as in the Example. Subsequent steps are: in situ histo-chemicalstaining method was used to detect the lacZ expression. The positiveresult was obtained in both male gametophytes and zygotic sporophytes.Transformation efficiency in the latter is to 70%, the integration oflacZ gene was suggested by positive PCR results.

The said functional foreign genes in this invention also include thoseencoding phycobillisome, growth factor, antibody or antigen; Saidantibiotics or herbicide resistance genes also include bar gene; Saidherbicide also include Basta; Said promoters also include CaMV35Spromoter or ubiquitin promoter; Said selectable marker-selectablereagent combinations also include bar-Basta; Said reporter gene alsoinclude luc gene or GFP gene.

1. A method to establish transgenic seaweeds, comprising the followingsteps: Constructing the vector for transformation by inserting thehigh-plant or algae-derived promoters upstream of foreign reporter genesor such cassettes that functional genes are fused with antibiotics orherbicide-resistant genes; Introducing said recombinated plasmid DNA toseaweed spore with Biolostics as transformation methods; Generating thegenetic seaweed through natural development process.
 2. The method toestablish transgenic seaweeds according to the claim 1, characterized inthat wherein introduced traits of antibiotics or herbicide-resistancewere used to select the transgenic seaweed individuals when foreignfunctional genes are transformed.
 3. The method to establish transgenicseaweeds according to the claim 1 and 2, characterized in that whereinsaid foreign functional genes include those encoding phycobillisome,growth factor, antibody, vaccine, denfensin or antibacterial peptide. 4.The method to establish transgenic seaweeds according to the claim 1 and2, characterized in that wherein said promoters include SV40 promoter,CaMV35S promoter, ubiquitin promoter or algae-derived fcp promoter. 5.The method to establish transgenic seaweeds according to the claim 1 and2, characterized in that wherein said resistance genes include cat gene,hpt gene or bar gene.
 6. The process of claim 1 and 2, wherein saidantibiotics include chloramphenicol or hygromycin.
 7. The method toestablish transgenic seaweeds according to the claim 1 and 2,characterized in that wherein said herbicide is Basta.
 8. The method toestablish transgenic seaweeds according to the claim 1 and 2,characterized in that wherein said transformation methods includeBiolistics or electroporation.
 9. The method to establish transgenicseaweeds according to the claim 1 and 2, characterized in that whereinsaid selectable marker gene-selection reagent combinations include catgene-chloramphenicol, hpt gene-hygromycin or bar gene-Basta.
 10. Themethod to establish transgenic seaweeds according to the claim 1,characterized in that wherein said foreign reporter genes include GUSgene, lacZ gene, luc gene or GFP gene.